Gene Expression Analysis of Conjunctival Inflammation

After 5 days of DED induction, tissues of the conjunctival sac, including bulbar conjunctiva and fornical conjunctiva, were collected; the conjunctivae of four eyes were pooled together as a sample. RNA was extracted using Nucleospin RNA kits (Thermo Fisher Scientific, Waltham, MA, USA) and then reverse transcribed into cDNA using the PrimeScript First-strand cDNA synthesis kit (TaKaRa, Dalian, China). RT-PCR was performed using SYBR Green reagents (R&D) and predesigned primers for IL-17A, IFN-γ, IL-1β, and IL-6. The samples were analyzed using Bio-Rad CFX Maestro1.1 (Bio-Rad Laboratories, Philadelphia, PA, USA). The quantification data were analyzed as described previously³⁵ (link) using GAPDH as an internal control.